ORGANIZATION, EXPRESSION, AND INHERITANCE OF YEAST MTDNA

点击翻译按钮获取中文摘要

The long term goals of the proposed research are 1) to understand the bifunctional nature of a novel protein (p3l) encoded by intron 4 of the yeast mitochondrial COXr gene (aI4alpha) that has both a DNA endonuclease activity required for intron homing and a latent maturase (splicing) activity; and 2) to understand how yeast cells organize and regulate the copy number of their mitochondrial DNA (mtDNA) so as to transmit mtDNA to progeny cells and insure a respiratory competent state. In the previous grant period we identified separate critical domains of p3l that are required for its endonuclease activity and for its maturase function. A key element of goal I of the current proposal is to test the hypothesis that activation of the latent maturaSe activity of p3l requires a productive interaction with another bifunctional protein, Nam2p, encoded by the nuclear nam2 gene that is both a mitochondrial leucyl tRNA synthetase and a splicing factor. These studies of p3l and Nam2p are aimed at an in vitro reconstitution of protein-dependent splicing of aI4alpha. The second goal will exploit the -finding that the HMO-like mitochondrial protein, Abf2p, is required for the maintenance of wild-type (p+) mtDNA and appears to be involved in mtDNA copy control. We will characterize spontaneous suppressors of an abf2 null mutant and an unexpected multicopy suppressor gene, ILV5, which we identified that encodes a mitochondrial protein (AHAR) that catalyzes a step in the biosynthesis of Ile, Leu and Val. We will test the hypothesis that AHAR is a bifunctional protein required for the synthesis of branched chain amino acids and for mtDNA stability. Finally, we will carry out a systematic biochemical and genetic analysis of the proteins associated with mtDNA nucleoids to correlate the organizational properties mtDNA-protein complexes with mtDNA maintenance. The ultimate objective of these studies is to understand the mechanisms of mtDNA segregation and copy number control and to provide insight into the putative mtDNA segregation apparatus. The issue of how cells transmit and control the amount of their mtDNA is fundamental and applies to almost all eukaryotic cells but is poorly understood. It is now clear that a number of inherited human disorders involve aspects of the segregation and copy number of mutant and wild-type mtDNAs. We have developed a tractable experimental system in which to investigate these important issues.